Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

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• (46)Deli J., and Warren G.F., 1971 ‘Adsorption, desorption and leaching of diphenamid in soils’. Weed Sci. 19, p. 67-69.

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• (49)Pionke H.B., and Deangelis R.J., (1980) ‘Methods for distributing pesticide loss in field run-off between the solution and adsorbed phase’, CREAMS, in A Field Scale Model for Chemicals, Run-off and Erosion from Agricultural Management Systems, Chapter 19, Vol. III: Supporting Documentation, USDA Conservation Research report.

• (50)ISO Standard Compendium Environment: Soil Quality — General aspects; chemical and physical methods of analysis; biological methods of analysis. First Edition (1994).

• (51)Scheffer F., and Schachtschabel, Lehrbuch der Bodenkunde, F. Enke Verlag, Stuttgart (1982) 11th edition.

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• (53)ISO/DIS 10381-1 Soil Quality — Sampling — Part 1: Guidance on the design of sampling programmes.

• (54)ISO/DIS 10381-2 Soil Quality — Sampling — Part 2: Guidance on sampling techniques.

• (55)ISO/DIS 10381-3 Soil Quality — Sampling — Part 3: Guidance on safety of sampling.

• (56)ISO/DIS 10381-4 Soil Quality — Sampling — Part 4: Guidance on the investigation of natural and cultivated soils.

• (57)ISO/DIS 10381-5 Soil Quality — Sampling — Part 5: Guidance on the investigation of soil contamination of urban and industrial sites.

• (58)ISO 10381-6, 1993: Soil Quality — Sampling — Part 6: Guidance on the collection, handling and storage of soil for the assessment of aerobic microbial processes in the laboratory.

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• (62)Boesten, J.J.T.I. ‘Influence of soil/liquid ratio on the experimental error of sorption coefficients in relation to OECD guideline 106’ Proceedings of 5th international workshop on environmental behaviour of pesticides and regulatory aspects, Brussels, 26-29 April 1994.

• (63)Bastide J., Cantier J.M., et Coste C., (1980) ‘Comportement de substances herbicides dans le sol en fonction de leur structure chimique’. Weed Res. 21, p. 227-231.

• (64)Brown D.S., and Flagg E.W., (1981) ‘Empirical prediction of organic pollutants sorption in natural sediments’. J. Environ.Qual., 10(3), p. 382-386.

• (65)Chiou C.T., Porter P.E., and Schmedding D.W., (1983) ‘Partition equilibria of non-ionic organic compounds between soil organic matter and water’. Environ. Sci. Technol., 17(4), p. 227-231.

• (66)Gerstl Z., and Mingelgrin U., (1984) ‘Sorption of organic substances by soils and sediments’. J. Environm. Sci. Health, B19 (3), p. 297-312.

• (67)Vowles P.D., and Mantoura R.F.C., (1987), ‘Sediment-water partition coefficient and HPLC retention factors of aromatic hydrocarbons’. Chemosphere, 16(1), p. 109-116.

• (68)Lyman W.J., Reehl W.F.and Rosenblatt D.H. (1990) Handbook of Chemical Property Estimation Methods. Environmental Behaviour of Organic Compounds. American Chemical Society, Washington DC.

• (69)Keniga E.E., and Goring, C.A.I. (1980) ‘Relationship between water solubility, soil sorption, octanol-water partitioning and concentration of chemicals in the biota’ in Aquatic Toxicology (eds J.G. Eaton, et al.), p. 78-115, ASTM STP 707, Philadelphia.

• (70)Chiou C.T., Peters L.J., and Freed V.H., (1979) ‘A physical concept of soil-water equilibria for non-ionic organic compounds’. Science, Vol. 206, p. 831-832.

• (71)Hassett J.J., Banwart W.I., Wood S.G., and Means J.C., (1981) ‘Sorption of/-Naphtol: implications concerning the limits of hydrophobic sorption’. Soil Sci. Soc. Am. J. 45, p. 38-42.

• (72)Karickhoff S.W., (1981), ‘Semi-empirical estimation of sorption of hydrophobic pollutants on natural sediments and soils’. Chemosphere, Vol. 10(8), p. 833-846.

• (73)Moreale A., van Bladel R., (1981) ‘Adsorption de 13 herbicides et insecticides par le sol. Relation solubilité — reactivité’. Revue de l'Agric., 34 (4), p. 319-322.

• (74)Müller M., Kördel W. (1996) ‘Comparison of screening methods for the determination/estimation of adsorption coefficients on soil’. Chemosphere, 32(12), p. 2493-2504.

• (75)Kördel W., Kotthoff G., Müller M. (1995) ‘HPLC — screening method for the determination of the adsorption coefficient on soil — results of a ring test’. Chemosphere 30 (7), p. 1373-1384.

• (76)Kördel W., Stutte J., Kotthoff G. (1993), 'HPLC — screening method for the determination of the adsorption coefficient on soil — comparison of different stationary phases. Chemosphere 27 (12), p. 2341-2352.

• (77)Hance, R.J., (1967), ‘The speed of Attainment of Sorption Equilibria in Some Systems Involving Herbicides’. Weed Research, Vol. 7, p. 29-36.

• (78)Koskinen W.C., and Harper S.S., (1990), ‘The retention processes: mechanisms’ in Pesticides in the Soil Environment: Processes, Impacts and Modelling (ed. H.H. Cheng). Soil Sci. Soc. Am. Book Series, No 2, Madison, Wisconsin.

• (79)Cohen S.Z., Creeger S.M., Carsel R.F., and Enfield C.G. (1984), ‘Potential pesticide contamination of groundwater from agricultural uses’, in Treatment and Disposal of Pesticide Wastes, p. 297-325, ACS Symp. Ser. 259, American Chemical Society, Washington, DC.

• (80)Giles C.H., (1970) ‘Interpretation and use of sorption isotherms’ in Sorption and Transport Processes in Soils. S.C.I. Monograph No. 37, p. 14-32.

• (81)Giles, C.H.; McEwan J.H.; Nakhwa, S.N. and Smith, D, (1960) ‘Studies in adsorption: XI. A system of classification of solution adsorption isotherms and its use in the diagnosis of adsorption mechanisms and in measurements of pesticides surface areas of soils’. J. Chem. Soc., p. 3973-93.

• (82)Calvet R., Tercé M., and Arvien J.C., (1980) ‘Adsorption des pesticides par les sols et leurs constituants: 3. Caractéristiques générales de l'adsorption’. Ann. Agron. 31, p. 239-251.

• (83)Bedbur E., (1996) ‘Anomalies in the Freundlich equation’, Proc. COST 66 Workshop, Pesticides in soil and the environment, 13-15 May 1996, Stratford-upon-Avon, U.K.

• (84)Guth, J.A., (1985) ‘Adsorption/desorption’, in Joint International Symposium, Physicochemical Properties and their Role in Environmental Hazard Assessment, July 1-3, Canterbury, UK.

• (85)Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1 (1985) and Soil Sci. Soc. Amer. Proc. 26, p. 305 (1962).

Appendix 1

Testing scheme

Image omitted; see official source

Text of image

Set up of a suitable analytical method

Available?

Yes

No

No further test

Prerequisite

Preliminary study

2 Soils

Selection of optimal soil/solution ratios

Determination of equilibrium time

Check for adsorption on the vessel surface

Check for the stability by means of parental mass balance

Tier 1

Equilibrium plateau is achieved?

Yes

Adsorption kinetics

Tier 2

If mass balance < 90 %, test substance is unstable in the time scale of the test. The test test can continue by analysing both phases (soil and aqueous solution).

5 Soils

Kd*(msoi/V0) > 0.3 (indirect method: measurement of solution depletion)

Kd*(msoi/V0) > 0.1 (direct method: measurement of both phases)

as appropriate

Tier 3

Adsorption isotherms

Desorption kinetics

Desorption isotherms

Appendix 2

INFLUENCE OF ACCURACY OF ANALYTICAL METHOD AND CONCENTRATION CHANGE ON ACCURACY OF ADSORPTION RESULTS

From the following table (84) it becomes obvious that when the difference between the initial mass (m0 = 110 μg) and equilibrium mass (

Image omitted; see official source = 100 μg) of the test substance in the solution is very small, an error of 5 % in the measurement of equilibrium concentration results in an error of 50 % in the calculation of the mass of the substance adsorbed in soil (

Image omitted; see official source ) and of 52,4 % in the calculation of the Kd.

Amount of soil	msoil	= 10 g
Volume of solution	V0	= 100 cm3
	Image omitted; see official source (μg)	Image omitted; see official source (μg cm-3)
---	---	---
	FOR A = 9 %	FOR A = 9 %
m0 = 110 μg or C0 = 1,100 μg/cm3	100	1,000
m0 = 110 μg or C0 = 1,100 μg/cm3	101	1,010
m0 = 110 μg or C0 = 1,100 μg/cm3	105	1,050
m0 = 110 μg or C0 = 1,100 μg/cm3	109	1,090
	FOR A = 55 %	FOR A = 55 %
m0 = 110 μg or C0 = 1,100 μg/cm3	50,0	0,500
m0 = 110 μg or C0 = 1,100 μg/cm3	50,5	0,505
m0 = 110 μg or C0 = 1,100 μg/cm3	52,5	0,525
m0 = 110 μg or C0 = 1,100 μg/cm3	55,0	0,550
	FOR A = 99 %	FOR A = 99 %
m0 = 110 μg or C0 = 1,100 μg/cm3	1,100	0,011
m0 = 110 μg or C0 = 1,100 μg/cm3	1,111	0,01111
m0 = 110 μg or C0 = 1,100 μg/cm3	1,155	0,01155
m0 = 110 μg or C0 = 1,100 μg/cm3	1,21	0,0121
---	---	---
Image omitted; see official source	=	Image omitted; see official source
---	---	---
Image omitted; see official source	=	mass of the test substance in the soil phase at equilibrium, μg;
---	---	---
Image omitted; see official source	=	mass of the test substance in the aqueous phase at equilibrium, μg;
---	---	---
Image omitted; see official source	=	content of the test substance in the soil phase at equilibrium, μg g-1;
---	---	---
Image omitted; see official source	=	mass concentration of the test substance in the aqueous phase at equilibrium, μg cm-3;
---	---	---
R	=	analytical error in the determination of the Image omitted; see official source;
---	---	---
R‡	=	calculated error due to the analytical error R.

Appendix 3

ESTIMATION TECHNIQUES FOR KD

• 1.Estimation techniques permit prediction of Kd based on correlations with, for example, Pow values (12)(39)(63-68), water solubility data (12)(19)(21)(39)(68-73), or polarity data derived by application of HPLC on reversed phase (74-76). As shown in Tables 1 and 2, is the Koc or Kom that are calculated from these equations and then, indirectly, the Kd from the equations:

Image omitted; see official source	Image omitted; see official source

• 2.The concept of these correlations is based on two assumptions: (1) it is the organic matter of the soil that mainly influences the adsorption of a substance; and (2) the interactions involved are mainly non-polar. As a result, these correlations: (1) are not, or are only to some extent, applicable to polar substances, and (2) are not applicable in cases where the organic matter content of the soil is very small (12). In addition, although satisfactory correlations have been found between Pow and adsorption (19), the same cannot be said for the relationship between water solubility and extent of adsorption (19)(21); so far the studies are very contradictory.

• 3.Some examples of correlations between the adsorption coefficient and the octanol-water partition coefficient, as well as water solubility are given in Tables 1 and 2, respectively.

Table 1

Examples of correlations between the adsorption distribution coefficient and the octanol-water partition coefficient; for further examples (12) (68).

Substances	Correlations	Authors
Substituted ureas	log Kom = 0,69 + 0,52 log Pow	Briggs (1981) (39)
Aromatic chlorinated	log Koc = - 0,779 + 0,904 log Pow	Chiou et al. (1983) (65)
Various pesticides	log Kom = 4,4 + 0,72 log Pow	Gerstl and Mingelgrin (1984) (66)
Aromatic hydrocarbons	log Koc = - 2,53 + 1,15 log Pow	Vowles and Mantoura (1987) (67)

Table 2

Examples of correlations between the adsorption distribution coefficient and water solubility; for further examples see (68) (69).

Compounds	Correlations	Authors
Various pesticides	log Kom = 3,8 - 0,561 log Sw	Gerstl and Mingelgrin (1984) (66)
Aliphatic, aromatic chlorinated substances	log Kom = (4,040 +/- 0,038 ) - (0,557 +/- 0,012 ) log Sw	Chiou et al. (1979) (70)
α-naphtol	logKoc = 4,273 - 0,686 log Sw	Hasset et al. (1981) (71)
Cyclic, aliphatic aromatic substances	logKoc = - 1,405 - 0,921 log Sw - 0,00953 (mp-25)	Karickhoff (1981) (72)
Various compounds	log Kom = 2,75 - 0,45 log Sw	Moreale van Blade (1982) (73)

Appendix 4

CALCULATIONS FOR DEFINING THE CENTRIFUGATION CONDITIONS

• 1.The centrifugation time is given by the following formula, assuming spherical particles:

Image omitted; see official source	(1)

• For simplification purposes, all parameters are described in non-SI units (g, cm).

• where:

• ω = rotational speed (=2 π rpm/60), rad s^-1

• rpm = revolutions per minute

• η = viscosity of solution, g s^-1 cm^-1

• rp = particle radius, cm

• ρs = soil density, g cm^-3

• ρaq = solution density, g cm^-3

• Rt = distance from the centre of centrifuge rotor to top of solution in centrifuge tube, cm

• Rb = distance from the centre of centrifuge rotor to bottom in centrifuge tube, cm

• Rb-Rt = length of the soil/solution mixture in the centrifuge tube, cm.

• In general practice, double the calculated times is used to ensure complete separation.

• 2.The equation (1) can be simplified further if we consider the viscosity (η) and the density (ρaq) of the solution as equal to the viscosity and density of water at 25 ^oC; thus, η = 8,95 × 10^-3 g s^-1 cm^-1 and ρaq = 1,0 g. cm^-3.

• Then, the centrifugation time is given by the equation (2):

Image omitted; see official source	(2)

• 3.From the equation (2) it becomes apparent that two parameters are important in defining the centrifugation condition, i.e. time (t) and speed (rpm), in order to achieve separation of particles with a specific size (in our case 0,1 μm radius): (1) the density of the soil and (2) the length of the mixture in the centrifuge tube (Rb-Rt), i.e. the distance which a soil particle covers from the top of the solution to the bottom of the tube; obviously, for a fixed volume the length of the mixture in the tube will depend on the square of the radius of the tube.

• 4.Fig. 1 presents variations in the centrifugation time (t) versus centrifugation speed (rpm) for different soil densities (ρs) (Fig.1a) and different lengths of the mixture in the centrifuge tubes (Fig.2a). From Fig.1a the influence of the soil density appears obvious; for example, for a classical centrifugation of 3 000 rpm the centrifugation time is approximately 240 min for 1,2 g cm³ soil density, while it is only 50 min for 2,0 g cm³. Similarly, from Fig 1b, for a classical centrifugation of 3 000 rpm the centrifugation time is approximately 50 min for a length of the mixture of 10 cm and only seven min for a length of 1 cm. However, it is important to find an optimal relation between centrifugation which requires the less length possible and easy handling for the experimenter in separating the phases after centrifugation.

• 5.Moreover, when defining the experimental conditions for the separation of soil/solution phases, it is important to consider the possible existence of a third ‘pseudo-phase’, the colloids. These particles, with a size less than 0,2 μm, can have an important impact on the whole adsorption mechanism of a substance in a soil suspension. When centrifugation is performed as described above, colloids remain in the aqueous phase and are subjected to analysis together with the aqueous phase. Thus, the information about their impact is lost.

• If the conducting laboratory has ultracentrifugation or ultrafiltration facilities, the adsorption/desorption of a substance in soil could be studied more in depth, including information on the adsorption of the substance on the colloids. In this case, an ultracentrifugation at 60 000 rpm/min or an ultrafiltration with filter porosity of 100 000 Daltons should be applied in order to separate the three phases soil, colloids, solution. The test protocol should also be modified accordingly, in order all three phases to be subjected to substance analysis.

Fig. 1a.

Image omitted; see official source

Text of image

Centrifugation time (min)

Centrifugation speed (rpm)

1 day

1 hour

Variations of centrifugation time (t) versus centrifugation speed (rpm) for different soil densities (ρs). Rt = 10 cm, Rb - Rt = 10 cm, η = 8,95 × 10^-3 g s^-1 cm^-1 and ρaq = 1,0 g. cm^-3 at 25 ^oC.

Fig. 1b.

Image omitted; see official source

Text of image

Centrifugation time (min)

Centrifugation speed (rpm)

1 day

1 hour

Variations of centrifugation time (t) versus centrifugation speed (rpm) for different lengths of the mixture in the centrifuge tube (Rb - Rt) = L; Rt = 10 cm, η = 8,95 × 10^-3 g s^-1 cm^-1, ρaq = 1,0 g. cm^-3 at 25 ^oC and ρs = 2,0 g cm^-3.

Appendix 5

CALCULATION OF ADSORPTION A (%) AND DESORPTION D (%)

The time scheme of the procedure is:

Image omitted; see official source

Text of image

Time t

For all the calculations it is assumed that the test substance is stable and does not adsorb significantly to the container walls.

ADSORPTION A (A%)

a) Parallel method

The percentage adsorption is calculated for each test tube (i) at each time point (ti), according to the equation:

Image omitted; see official source (%)	(1)

The terms of this equation may be calculated as follows:

m0 = C0 · V0 (μg)	(2)
Image omitted; see official source (μg)	(3)

where:

Image omitted; see official source	=	adsorption percentage (%) at the time point ti
---	---	---
Image omitted; see official source	=	mass of the test substance on soil at the time ti that the analysis is performed (μg)
---	---	---
m0	=	mass of test substance in the test tube, at the beginning of the test (μg)
---	---	---
C0	=	initial mass concentration of the test solution in contact with the soil (μg cm-3)
---	---	---
Image omitted; see official source	=	mass concentration of the substance in the aqueous phase at the time ti that the analysis is performed (μg cm-3); this concentration is analytically determined taking into account the values given by the blanks
---	---	---
V0	=	initial volume of the test solution in contact with the soil (cm3).

The values of the adsorption percentage

Image omitted; see official source

or

Image omitted; see official source

are plotted versus time and the time after which the sorption equilibrium is attained is determined. Examples of such plots are given in Fig. 1 and Fig. 2 respectively.Image omitted; see official source

Text of image

Concentration C^ads aq (μg cm^-3)

C0

C^ads aq (eq)

Equilibration time ti (h)

Fig. 1.

Adsorption equilibrium plot

Image omitted; see official source

Fig. 2.

Mass concentration of the test substance in the aqueous phase (Caq) versus time

b) Serial method

The following equations take into account that the adsorption procedure is carried out by measurements of the test substance in small aliquots of the aqueous phase at specific time intervals.

During each time interval the amount of the substance adsorbed on the soil is calculated as follows:

• — for the first time interval Δt1 = t1 - t0 Image omitted; see official source (4)

• — for the second time interval Δt2 = t2 - t1 Image omitted; see official source (5)

• — for the third time interval Δt3 = t3 - t2 Image omitted; see official source (6)

• — for the nth time interval Δtn = tn - tn - 1 Image omitted; see official source (7)

The percentage of adsorption at each time interval,

Image omitted; see official source

, is calculated using the following equation:

Image omitted; see official source	(8)

while the percentage of adsorption

Image omitted; see official source

at a time point ti is given by the equation:

Image omitted; see official source	(9)

The values of the adsorption

Image omitted; see official source

or

Image omitted; see official source

(with respect to the needs of the study) are plotted versus time and the time after which the sorption equilibrium is attained is determined.

At the equilibration time teq:

• — the mass of the test substance adsorbed on the soil is: Image omitted; see official source (10)

• — the mass of the test substance in the solution is: Image omitted; see official source (11)

• — and the percentage of adsorption at equilibrium is: Image omitted; see official source (12)

The parameters used above are defined as:

Image omitted; see official source	=	mass of the substance adsorbed on the soil during the time intervals Δt1, Δt2,..., Δtn respectively (μg);
---	---	---
Image omitted; see official source	=	mass of the substance measured in an aliquot Image omitted; see official source at the time points t1, t2,tn respectively (μg);
---	---	---
Image omitted; see official source	=	mass of the substance adsorbed on the soil at adsorption equilibrium (μg);
---	---	---
Image omitted; see official source	=	mass of the substance in the solution at adsorption equilibrium (μg);
---	---	---
Image omitted; see official source	=	volume of the aliquot in which the test substance is measured (cm3);
---	---	---
Image omitted; see official source	=	percentage of adsorption corresponding at a time interval Δti (%);
---	---	---
Image omitted; see official source	=	percentage of adsorption at adsorption equilibrium (%).

DESORPTION D (A %)

The time t0 that the desorption kinetics experiment begins, is considered as the moment that the maximal recovered volume of the test substance solution (after that the adsorption equilibrium is attained) is replaced by an equal volume of 0,01 M CaCl2 solution.

(a) Parallel method

At a time point ti, the mass of the test substance is measured in the aqueous phase taken from the tube i (

Image omitted; see official source ), and the mass desorbed is calculated according to the equation:

Image omitted; see official source	(13)

At desorption equilibrium ti = teq and therefore

Image omitted; see official source =

Image omitted; see official source

The mass of the test substance desorbed during a time interval (Δti) is given by the equation:

Image omitted; see official source	(14)

The percentage of desorption is calculated:

at a time point ti from the equation:

Image omitted; see official source	(15)

and during a time interval (Δti) from the equation:

Image omitted; see official source	(16)

where:

Image omitted; see official source	=	desorption percentage at a time point ti (%)
---	---	---
Image omitted; see official source	=	desorption percentage corresponding to a time interval Δti (%)
---	---	---
Image omitted; see official source	=	mass of the test substance desorbed at a time point ti, (μg)
---	---	---
Image omitted; see official source	=	mass of the test substance desorbed during a time interval Δti (μg)
---	---	---
Image omitted; see official source	=	mass of the test substance analytically measured at a time ti in a solution volume Image omitted; see official source ,which is taken for the analysis (μg)
---	---	---
Image omitted; see official source	=	mass of the test substance left over from the adsorption equilibrium due to incomplete volume replacement (μg)
---	---
Image omitted; see official source	(17)
---	---	---
Image omitted; see official source	=	mass of the test substance in the solution at adsorption equilibrium (μg)
---	---	---
VR	=	volume of the supernatant removed from the tube after the attainment of adsorption equilibrium and replaced by the same volume of a 0,01 M CaCl2 solution (cm3)
---	---	---
Image omitted; see official source	=	volume of the solution taken from the tube (i) for the measurement of the test substance, in desorption kinetics experiment (cm3).

The values of desorption

Image omitted; see official source

or

Image omitted; see official source

(according to the needs of the study) are plotted versus time and the time after which the desorption equilibrium is attained is determined.

(b) Serial method

The following equations take into account that the adsorption procedure, which was preceded, was carried out by measurement of test substance in small aliquots

Image omitted; see official source of the aqueous phase (serial method in ‘Performance of the test’ 1,9). It is assumed that: (a) the volume of the supernatant removed from the tube after the adsorption kinetics experiment was replaced by the same volume of 0,01 M CaCl2 solution (VR) and (b) and the total volume of the aqueous phase in contact with the soil (VT) during the desorption kinetics experiment remains constant and is given by the equation:

Image omitted; see official source	(18)

At a time point ti:

• — The mass of the test substance is measured in a small aliquot Image omitted; see official source and the mass desorbed is calculated, according to the equation: Image omitted; see official source (19)

• — At desorption equilibrium ti = teq and therefore Image omitted; see official source = Image omitted; see official source.

• — The percentage of desorption Image omitted; see official source is calculated, from the following equation: Image omitted; see official source (20)

At a time interval (Δti):

During each time interval the amount of the substance desorbed is calculated as follows:

• — for the first time interval Δt1 = t1-t0 Image omitted; see official source and Image omitted; see official source (21)

• — for the second time interval Δt2 = t2-t1 Image omitted; see official source andImage omitted; see official source (22)

• — for the n^th interval Δtn = tn-tn-1 Image omitted; see official source andImage omitted; see official source (23)

Finally, the percentage of desorption at each time interval,

Image omitted; see official source

, is calculated using the following equation:

Image omitted; see official source	(24)

while the percentage of desorption

Image omitted; see official source at a time point ti is given by the equation:

Image omitted; see official source	(25)

where the above used parameters are defined as:

Image omitted; see official source, Image omitted; see official source, ..., Image omitted; see official source	=	mass of the substance remaining adsorbed on the soil after the time intervals Δt1, Δt2, ..., Δtn respectively (μg)
---	---	---
Image omitted; see official source, Image omitted; see official source, ..., Image omitted; see official source	=	mass of the test substance desorbed during the time intervals Δt1, Δt2, ..., Δtn respectively (μg)
---	---	---
Image omitted; see official source, Image omitted; see official source, ..., Image omitted; see official source	=	mass of the substance measured in an aliquot Image omitted; see official source at time points t1,t2, ..., tn, respectively (μg)
---	---	---
VT	=	total volume of the aqueous phase in contact with the soil during the desorption kinetics experiment performed with the serial method (cm3)
---	---	---
Image omitted; see official source	=	mass of the test substance left over from the adsorption equilibrium due to incomplete volume replacement (μg) Image omitted; see official source (26)
---	---	---
VR	=	volume of the supernatant removed from the tube after the attainment of adsorption equilibrium and replaced by the same volume of a 0,01 M CaCl2 solution (cm3)
---	---	---
Image omitted; see official source	=	volume of the aliquot sampled for analytical purpose from the tube (i), during the desorption kinetics experiment performed with the serialmethod (cm3) Image omitted; see official source (27)

Appendix 6

ADSORPTION-DESORPTION IN SOILS: DATA REPORTING SHEETS

Substance tested:

Soil tested:

Dry mass content of the soil (105 ^oC, 12h): … %

Temperature: … ^oC

Suitability of the analytical method

Weighed soil	g
Soil: dry mass	g
Volume CaCl2 sol.	cm3
Nominal conc. final sol.	μg cm-3
Analytical conc. final sol.	μg cm-3

Principle of the analytical method used:

Calibration of the analytical method:

Substance tested:

Soil tested

Dry mass content of the soil (105 ^oC, 12 h): … %

Temperature: … ^oC

Analytical methodology followed:	Indirect		Parallel		Serial	
	Direct	

Adsorption test: test samples

	Symbol	Units	Equilibration time	Equilibration time	Equilibration time	Equilibration time	Equilibration time	Equilibration time	Equilibration time	Equilibration time
Tube No
Weighed soil	—	g
Soil: dry mass	msoil	g
Water volume in weighed soil (calculated)	VWS	cm3
Volume 0,01 M CaCl2 sol. to equilibrate the soil		cm3
Volume of stock solution		cm3
Total volume of aq. phase in contact with soil	V0	cm3
Initial concentration Test solution	C0	μg cm-3
Mass test subst. at the beginning of the test	m0	μg
After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation
INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD	INDIRECT METHOD
Parallel method	Parallel method	Parallel method	Parallel method	Parallel method	Parallel method	Parallel method	Parallel method	Parallel method	Parallel method	Parallel method
Concentration test subst. aq. phase Blank correction included	Image omitted; see official source	μg cm-3
Serial method	Serial method	Serial method	Serial method	Serial method	Serial method	Serial method	Serial method	Serial method	Serial method	Serial method
Measured mass test subst. in the aliquot Va A	Image omitted; see official source	μg
DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD	DIRECT METHOD
Mass test substance adsorbed on soil	Image omitted; see official source	μg
Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption	Calculation of adsorption
Adsorption	Image omitted; see official source	%
	Image omitted; see official source	%
Means
Adsorption coefficient	Kd	cm3 g-1
Means
Adsorption coefficient	Koc	cm3 g-1
Means

Substance tested:

Soil tested:

Dry mass content of the soil (105 ^oC, 12 h): … %

Temperature: … ^oC

Adsorption test: blanks and control

	Symbol	Units	Blank	Blank	Blank	Blank	Control	Control
Tube No
Weighed soils		g					0	0
Water amount in weighed soil (calculated)		cm3					—	—
Volume of 0,01 M CaCl2 solution added		cm3
Volume of the stock solution of the test substance added		cm3	0	0
Total volume of aq. phase (calculated)		cm3					—	—
Initial concentration of the test substance in aqueous phase		μg cm-3
After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation
Concentration in aqueous phase		μg cm-3

Remark: add columns if necessary

Substance tested:

Soil tested:

Dry mass content of the soil (105 ^oC 12 h): … %

Temperature: … ^oC

Mass balance

	Symbol	Units
Tube No
Weighed soil	—	g
Soil: dry mass	msoil	g
Water volume in weighed soil (calculated)	VWS	ml
Volume 0,01 M CaCl2 sol. to equilibrate the soil		ml
Volume of stock solution		cm3
Total volume of aq. phase in contact with soil	V0	cm3
Initial concentration test solution	C0	μg cm-3
Equilibration time	—	h
After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation
Concentr. test subst. aq. phase at adsorption equilibrium blank correction included	Image omitted; see official source	μg cm-3
Equalibration time	teq	h
1st dilution with solvent	1st dilution with solvent	1st dilution with solvent	1st dilution with solvent	1st dilution with solvent	1st dilution with solvent	1st dilution with solvent
Removed volume aq. phase	Vrec	cm3
Added volume of solvent	ΔV	cm3
1st extraction with solvent	1st extraction with solvent	1st extraction with solvent	1st extraction with solvent	1st extraction with solvent	1st extraction with solvent	1st extraction with solvent
Signal analysed in solvent	SE1	var.
Conc. test subst. in solvent	CE1	μg cm-3
Mass of substance extracted from soil and vessel walls	mE1	μg
2nd dilution with solvent	2nd dilution with solvent	2nd dilution with solvent	2nd dilution with solvent	2nd dilution with solvent	2nd dilution with solvent	2nd dilution with solvent
Removed volume of solvent	ΔVs	cm3
Added volume of solvent	ΔV'	cm3
2nd extraction with solvent	2nd extraction with solvent	2nd extraction with solvent	2nd extraction with solvent	2nd extraction with solvent	2nd extraction with solvent	2nd extraction with solvent
Signal analysed in solvent phase	SE2	var.
Conc. test subst. in solvent	CE2	μg cm-3
Mass of substance extracted from soil and vessel walls	mE2	μg
Total mass test subst. extracted in two steps	mE	μg
Mass balance	MB	%

Substance tested:

Soil tested:

Dry mass content of the soil (105 ^oC, 12 h): … %

Temperature: … ^oC

Adsorption isotherms

	Symbol	Units
Tube No
Weighed soil	—	g
Soil: dry mass	E	g
Water volume in weighed soil (calculated)	VWS	cm3
Volume 0,01 M CaCl2 sol. to equilibrate the soil		cm3
Volume of stock solution added		cm3
Total volume of aq. phase in contact with soil (calculated)	V0	cm3
Concentration solution	C0	μg cm-3
Equilibration time	—	h
After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation	After agitation and centrifugation
Concentration subst. aq. phase, blank correction included	Image omitted; see official source	μg cm-3
Temperature		oC
Adsorb. mass per unit soil	Image omitted; see official source	μg g-1

Regression analysis:

value of KF ^ads:

value of l/n:

regression coefficient r²:

Substance tested:

Soil tested:

Dry mass content of the soil (105 ^oC, 12 h): … %

Temperature: … ^oC

Analytical methodology followed:	Indirect		Parallel		Serial	

Desorption test

		Symbol	Units	Time interval	Time interval	Time interval	Time interval
Tube No coming from adsorption step	Tube No coming from adsorption step
Mass of substance adsorbed on soil at adsorption equilibrium	Mass of substance adsorbed on soil at adsorption equilibrium	Image omitted; see official source	μg
Removed volume aq. phase, replaced by 0,01 M CaCl2	Removed volume aq. phase, replaced by 0,01 M CaCl2	VR	cm3
Total volume of aq. phase in contact with soil	PM	V0	cm3
Total volume of aq. phase in contact with soil	SM	VT	cm3
Mass test subst. left over the adsorption equilibrium due to incomplete volume replacement	Mass test subst. left over the adsorption equilibrium due to incomplete volume replacement	Image omitted; see official source	μg
Desorption kinetics	Desorption kinetics	Desorption kinetics	Desorption kinetics	Desorption kinetics	Desorption kinetics	Desorption kinetics	Desorption kinetics
Measured mass of substance desorbed from soil at time ti	Measured mass of substance desorbed from soil at time ti	Image omitted; see official source	μg
Volume of the solution taken from the tube (i) for the measurement of the test substance	PM	Vr i	cm3
Volume of the solution taken from the tube (i) for the measurement of the test substance	SM	va D	cm3
Mass of substance desorbed from soil at time ti (calculated)	Mass of substance desorbed from soil at time ti (calculated)	Image omitted; see official source	μg
Mass of substance desorbed from soil during time interval Δti (calculated)	Mass of substance desorbed from soil during time interval Δti (calculated)	Image omitted; see official source	μg
Desorption percentage	Desorption percentage	Desorption percentage	Desorption percentage	Desorption percentage	Desorption percentage	Desorption percentage	Desorption percentage
Desorption at time ti	Desorption at time ti	Dti	%
Desorption at time interval Δti	Desorption at time interval Δti	Image omitted; see official source	%
Apparent desorption coefficient	Apparent desorption coefficient	Kdes

PM: parallel method

SM: serial method

C.19.   ESTIMATION OF THE ADSORPTION COEFFICIENT (KOC) ON SOIL AND ON SEWAGE SLUDGE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

1. METHOD

This method is a replicate of OECD TG121 (2001).

1.1.   INTRODUCTION

The sorption behaviour of substances in soils or sewage sludges can be described through parameters experimentally determined by means of the test method C.18. An important parameter is the adsorption coefficient which is defined as the ratio between the concentration of the substance in the soil/sludge and the concentration of the substance in the aqueous phase at adsorption equilibrium. The adsorption coefficient normalised to the organic carbon content of the soil Koc is a useful indicator of the binding capacity of a chemical on organic matter of soil and sewage sludge and allows comparisons to be made between different chemicals. This parameter can be estimated through correlations with the water solubility and the n-octanol/water partition coefficient (1)(2)(3)(4)(5)(6)(7).

The experimental method described in this test uses HPLC for the estimation of the adsorption coefficient Koc in soil and in sewage sludge (8). The estimates are of higher reliability than those from QSAR calculations (9). As an estimation method it cannot fully replace batch equilibrium experiments used in the test method C18. However, the estimated Koc may be useful for choosing appropriate test parameters for adsorption/desorption studies according to the test method C.18 by calculating Kd (distribution coefficient) or Kf (Freundlich adsorption coefficient) according to the equation 3 (see Section 1.2).

1.2.   DEFINITIONS

K d: distribution coefficient is defined as the ratio of equilibrium concentrations C of a dissolved test substance in a two phase system consisting of a sorbent (soil or sewage sludge) and an aqueous phase; it is a dimensionless value when concentrations in both phases are expressed on a weight/weight base. In case the concentration in the aqueous phase is given on a weight/volume base then the units are ml· g^-1. Kd can vary with sorbent properties and can be concentration dependent.

Image omitted; see official source	(1)

where:

Csoil	=	concentration of test substance in soil at equilibrium (μg· g-1)
---	---	---
Csludge	=	concentration of test substance in sludge at equilibrium (μg· g-1)
---	---	---
Caq	=	concentration of test substance in aqueous phase at equilibrium (μg· g-1, μg· ml-1).

K f: Freundlich adsorption coefficient is defined as the concentration of the test substance in soil or sewage sludge (x/m) when the equilibrium concentration Caq in the aqueous phase is equal to one; units are μg.g^-1 sorbent. The value can vary with sorbent properties.

Image omitted; see official source	(2)

where:

x/m	=	amount of test substance x (μg) adsorbed on amount of sorbent m (g) at equilibrium
---	---	---
1/n	=	slope of Freundlich adsorption isotherm
---	---	---
Caq	=	concentration of test substance in aqueous phase at equilibrium (μg· ml- 1)

At

Image omitted; see official source

K oc: distribution coefficient (Kd) or Freundlich adsorption coefficient (Kf) normalised to the organic carbon content (foc) of a sorbent; particularly for non-ionised chemicals, it is an approximate indicator for the extent of adsorption between a substance and the sorbent and allows comparisons to be made between different chemicals. Depending on the dimensions of Kd and Kf, Koc can be dimensionless or have the units ml· g^-1 or μg· g^-1 organic matter.

Image omitted; see official source	(3)

The relationship between Koc and Kd is not always linear and thus Koc values can vary from soil to soil but their variability is greatly reduced compared to Kd or Kf values.

The adsorption coefficient (Koc) is deduced from the capacity factor (k') using a calibration plot of log k' versus log Koc of the selected reference compounds.

Image omitted; see official source	(4)

where:

tR	=	HPLC retention time of test and reference substance (minutes);
---	---	---
t0	=	HPLC dead time (minutes) (see Section 1.8.2).

POW: The octanol-water partition coefficient is defined as the ratio of the concentrations of dissolved substance in n-octanol and water; it is a dimensionless value.

Image omitted; see official source	(5)

1.3.   REFERENCE SUBSTANCES

The structural formula, the purity and the dissociation constant (if appropriate) should be known before using the method. Information on solubility in water and organic solvents, on octanol-water partition coefficient and on hydrolysis characteristics is useful.

To correlate the measured HPLC-retention data of a test substance with its adsorption coefficient Koc, a calibration graph of log Koc versus log k' has to be established. A minimum of six reference points, at least one above and one below the expected value of the test substance should be used. The accuracy of the method will be significantly improved if reference substances that are structurally related to the test substance are used. If such data are not available, it is up to the user to select the appropriate calibration substances. A more general set of structurally heterogeneous substances should be chosen in this case. Substances and Koc-values which may be used are listed in the Appendix in Table 1 for sewage sludge and in Table 3 for soil. The selection of other calibration substances should be justified.

1.4.   PRINCIPLE OF THE TEST METHOD

HPLC is performed on analytical columns packed with a commercially available cyanopropyl solid phase containing lipophilic and polar moieties. A moderately polar stationary phase based on a silica matrix is used:

— O — Si	— CH2 — CH2 — CH2	— CN
silica	non-polar spacer	polar moiety

The principle of the test method is similar to testing method A.8 (Partition coefficient, HPLC method). While passing through the column along with the mobile phase the test substance interacts with the stationary phase. As a result of partitioning between mobile and stationary phases the test substance is retarded. The dual composition of the stationary phase having polar and non-polar sites allows for interaction of polar and non-polar groups of a molecule in a similar way as is the case for organic matter in soil or sewage sludge matrices. This enables the relationship between the retention time on the column and the adsorption coefficient on organic matter to be established.

pH has a significant influence on sorption behaviour in particular for polar substances. For agricultural soils or tanks of sewage treatment plants pH normally varies between pH 5,5 and 7,5 . For ionisable substances, two tests should be performed with both ionised and non-ionised forms in appropriate buffer solutions but only in cases where at least 10 % of the test compound will be dissociated within pH 5,5 to 7,5 .

Since only the relationship between the retention on the HPLC column and the adsorption coefficient is employed for the evaluation, no quantitative analytical method is required and only the determination of the retention time is necessary. If a suitable set of reference substances is available and standard experimental conditions can be used, the method provides a fast and efficient way to estimate the adsorption coefficient Koc.

1.5.   APPLICABILITY OF THE TEST

The HPLC method is applicable to chemical substances (unlabelled or labelled) for which an appropriate detection system (e.g. spectrophotometer, radioactivity detector) is available and which are sufficiently stable during the duration of the experiment. It may be particularly useful for chemicals which are difficult to study in other experimental systems (i.e. volatile substances; substances which are not soluble in water at a concentration which can be measured analytically; substances with a high affinity to the surface of incubation systems). The method can be used for mixtures which give unresolved elution bands. In such a case, upper and lower limits of the log Koc values of the compounds of the test mixture should be stated.

Impurities may sometimes cause problems for interpretation of HPLC results, but they are of minor importance as long as the test substance can analytically be clearly identified and separated from the impurities.

The method is validated for the substances listed in Table 1 in the Appendix and was also applied to a variety of other chemicals belonging to the following chemical classes:

• — aromatic amines (e.g. trifluralin, 4-chloroaniline, 3,5-dinitroaniline, 4-methylaniline, N-methylaniline, 1-naphthylamine),

• — aromatic carboxilic acid esters (e.g. benzoic acid methylester, 3,5-dinitrobenzoic acid ethylester),

• — aromatic hydrocarbons (e.g. toluene, xylene, ethylbenzene, nitrobenzene),

• — aryloxyphenoxypropionic acid esters (e.g. diclofop-methyl, fenoxaprop-ethyl, fenoxaprop-P-ethyl),

• — benzimidazole and imidazole fungicides (e.g. carbendazim, fuberidazole, triazoxide),

• — carboxilic acid amides (e.g. 2-chlorobenzamide, N,N-dimethylbenzamide, 3,5-dinitrobenzamide, N-methylbenzamide, 2-nitrobenzamide, 3-nitrobenzamide),

• — chlorinated hydrocarbons (e.g. endosulfan, DDT, hexachlorobenzene, quintozene, 1,2,3-trichlorobenzene),

• — organophosphorus insecticides (e.g. azinphos-methyl, disulfoton, fenamiphos, isofenphos, pyrazophos, sulprofos, triazophos),

• — phenols (e.g. phenol, 2-nitrophenol, 4-nitrophenol, pentachlorophenol, 2,4,6-trichlorophenol, 1-naphthol),

• — phenylurea derivatives (e.g. isoproturon, monolinuron, pencycuron),

• — pigment dyestuffs (e.g. Acid Yellow 219, Basic Blue 41, Direct Red 81),

• — polyaromatic hydrocarbons (e.g. acenaphthene, naphthalene),

• — 1,3,5-triazine herbicides (e.g. prometryn, propazine, simazine, terbutryn),

• — triazole derivatives (e.g. tebuconazole, triadimefon, tradimenol, triapenthenol).

The method is not applicable for substances which react either with the eluent or the stationary phase. It is also not applicable for substances that interact in a specific way with inorganic components (e.g. formation of cluster complexes with clay minerals). The method may not work for surface active substances, inorganic compounds and moderate or strong organic acids and bases. Log Koc values ranging from 1,5 to 5,0 can be determined. Ionisable substances must be measured using a buffered mobile phase, but care has to be taken to avoid precipitation of buffer components or test substance.

1.6.   QUALITY CRITERIA

1.6.1.   Accuracy

Normally, the adsorption coefficient of a test substance can be estimated to within +/- 0,5 log unit of the value determined by the batch equilibrium method (see Table 1 in the Appendix). Higher accuracy may be achieved if the reference substances used are structurally related to the test substance.

1.6.2.   Repeatability

Determinations should be run at least in duplicate. The values of log Koc derived from individual measurements should be within a range of 0,25 log unit.

1.6.3.   Reproducibility

Experience gained so far in the application of the method is supportive of its validity. An investigation of the HPLC method, using 48 substances (mostly pesticides) for which reliable data on Koc on soils were available gave a correlation coefficient of R = 0,95 (10) (11).

An inter-laboratory comparison test with 11 participating laboratories was performed to improve and validate the method (12). Results are given in Table 2 of the Appendix.

1.7.   DESCRIPTION OF THE TEST METHOD

1.7.1.   Preliminary Estimation of the Adsorption Coefficient

The octanol-water partition coefficient Pow (= Kow) and, to some extent, the water solubility can be used as indicators for the extent of adsorption, particularly for non-ionised substances, and thus may be used for preliminary range finding. A variety of useful correlations have been published for several groups of chemicals (1)(2)(3)(4)(5)(6)(7).

1.7.2.   Apparatus